scholarly journals Quantification of the Detrimental Effect of a Single Primer-Template Mismatch by Real-Time PCR Using the 16S rRNA Gene as an Example

2008 ◽  
Vol 74 (5) ◽  
pp. 1660-1663 ◽  
Author(s):  
D. Bru ◽  
F. Martin-Laurent ◽  
L. Philippot
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy Number ◽  
Pcr Amplification ◽  
Gene Copy ◽  
Rrna Gene ◽  
Template Dna ◽  
Single Primer ◽  
The 16S Rrna Gene ◽  
Extension Sequence

ABSTRACT We investigated the effects of internal primer-template mismatches on the efficiency of PCR amplification using the 16S rRNA gene as the model template DNA. We observed that the presence of a single mismatch in the second half of the primer extension sequence can result in an underestimation of up to 1,000-fold of the gene copy number, depending on the primer and position of the mismatch.

scholarly journals Detection of Mycoplasma genitalium by PCR Amplification of the 16S rRNA Gene

2003 ◽  
Vol 41 (1) ◽  
pp. 261-266 ◽  
Author(s):  
J. S. Jensen ◽  
M. B. Borre ◽  
B. Dohn
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Amplification ◽  
Mycoplasma Genitalium ◽  
Rrna Gene ◽  
The 16S Rrna Gene

scholarly journals Biodiversity of Denitrifying and Dinitrogen-Fixing Bacteria in an Acid Forest Soil

2002 ◽  
Vol 68 (8) ◽  
pp. 3818-3829 ◽  
Author(s):  
Christopher Rösch ◽  
Alexander Mergel ◽  
Hermann Bothe
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nitrite Reductase ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Cloning And Sequencing ◽  
Genetic Traits ◽  
Uncultured Bacteria ◽  
Pcr Products ◽  
The 16S Rrna Gene

ABSTRACT Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome cd 1-containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification. For each gene segment, diverse PCR products were characterized by cloning and sequencing. None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium. In contrast, the analyzed clones of nifH gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N2-fixing bacterial population in this soil. Identical nifH sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area. Whereas biodiversity was high in the case of nosZ, only a few different sequences were obtained with nirK. With respect to nirS, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria. With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the Acidobacterium phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for nifH and for the denitrification genes were closely related to those of the proteobacteria. Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N2 fixation are not genetic traits of most of the uncultured bacteria.

scholarly journals Nonspecific PCR Amplification of the 16S rRNA Gene Segment in Different Bacteria by Use of Primers Specific for Campylobacter, Arcobacter, and Helicobacter spp.

2007 ◽  
Vol 45 (4) ◽  
pp. 1376-1377 ◽  
Author(s):  
A. D. Raut ◽  
B. P. Kapadnis ◽  
R. Shashidhar ◽  
J. R. Bandekar ◽  
P. Vaishampayan ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Segment ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
The 16S Rrna Gene

‘Candidatus Liberibacter americanus’, associated with citrus huanglongbing (greening disease) in São Paulo State, Brazil

2005 ◽  
Vol 55 (5) ◽  
pp. 1857-1862 ◽  
Author(s):  
Diva do Carmo Teixeira ◽  
Colette Saillard ◽  
Sandrine Eveillard ◽  
Jean Luc Danet ◽  
Paulo Inácio da Costa ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Intergenic Regions ◽  
The 16S Rrna Gene

Symptoms of huanglongbing (HLB) were reported in São Paulo State (SPS), Brazil, in March 2004. In Asia, HLB is caused by ‘Candidatus Liberibacter asiaticus' and in Africa by ‘Candidatus Liberibacter africanus’. Detection of the liberibacters is based on PCR amplification of their 16S rRNA gene with specific primers. Leaves with blotchy mottle symptoms characteristic of HLB were sampled in several farms of SPS and tested for the presence of liberibacters. ‘Ca. L. asiaticus' was detected in a small number of samples but most samples gave negative PCR results. Therefore, a new HLB pathogen was suspected. Evidence for an SPS-HLB bacterium in symptomatic leaves was obtained by PCR amplification with universal primers for prokaryotic 16S rRNA gene sequences. The amplified 16S rRNA gene was cloned and sequenced. Sequence analysis and phylogeny studies showed that the 16S rRNA gene possessed the oligonucleotide signatures and the secondary loop structure characteristic of the α-Proteobacteria, including the liberibacters. The 16S rRNA gene sequence phylogenetic tree showed that the SPS-HLB bacterium clustered within the α-Proteobacteria, the liberibacters being its closest relatives. For these reasons, the SPS-HLB bacterium is considered a member of the genus ‘Ca. Liberibacter’. However, while the 16S rRNA gene sequences of ‘Ca. L. asiaticus' and ‘Ca. L. africanus' had 98·4 % similarity, the 16S rRNA gene sequence of the SPS-HLB liberibacter had only 96·0 % similarity with the 16S rRNA gene sequences of ‘Ca. L. asiaticus' or ‘Ca. L. africanus’. This lower similarity was reflected in the phylogenetic tree, where the SPS-HLB liberibacter did not cluster within the ‘Ca. L asiaticus’/‘Ca. L. africanus group’, but as a separate branch. Within the genus ‘Candidatus Liberibacter’ and for a given species, the 16S/23S intergenic region does not vary greatly. The intergenic regions of three strains of ‘Ca. L. asiaticus’, from India, the People's Republic of China and Japan, were found to have identical or almost identical sequences. In contrast, the intergenic regions of the SPS-HLB liberibacter, ‘Ca. L. asiaticus' and ‘Ca. L. africanus' had quite different sequences, with similarity between 66·0 and 79·5 %. These results confirm that the SPS-HLB liberibacter is a novel species for which the name ‘Candidatus Liberibacter americanus' is proposed. Like the African and the Asian liberibacters, the ‘American’ liberibacter is restricted to the sieve tubes of the citrus host. The liberibacter could also be detected by PCR amplification of the 16S rRNA gene in Diaphorina citri, the psyllid vector of ‘Ca. L. asiaticus’, suggesting that this psyllid is also a vector of ‘Ca. L. americanus' in SPS. ‘Ca. L. americanus' was detected in 216 of 218 symptomatic leaf samples from 47 farms in 35 municipalities, while ‘Ca. L. asiaticus' was detected in only 4 of the 218 samples, indicating that ‘Ca. L. americanus' is the major cause of HLB in SPS.

Molecular identification of pufferfish species using PCR amplification and restriction analysis of a segment of the 16S rRNA gene

2006 ◽  
Vol 1 (1) ◽  
pp. 139-144 ◽  
Author(s):  
Shoichiro Ishizaki ◽  
Yasuhiro Yokoyama ◽  
Naomasa Oshiro ◽  
Natsuko Teruya ◽  
Yuji Nagashima ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Identification ◽  
Restriction Analysis ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
The 16S Rrna Gene
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